One approach to sequencing a large target nucleic acid, such as a human genome, is the use of shotgun sequencing. In shotgun sequencing, the target nucleic acid is fragmented or subcloned to produce a series of overlapping nucleic acid fragments and determining the sequence of these fragments. Based on the overlap and the knowledge of the sequence of each fragment, the complete sequence of a target nucleic acid can be constructed.
One disadvantage of the shotgun approach to sequencing is that assembly may be difficult if the target nucleic acid sequence comprise numerous small repeats (tandem or inverted repeats). The inability to assemble a genomic sequence in repeat regions leads to gaps in the assembled sequence. Thus, following initial assembly of a nucleic acid sequence, gaps in sequence coverage would need to be filled and uncertainties in assembly would need to be resolved.
One method of resolving these gaps is to use larger clones or fragments for sequencing because these larger fragments would be long enough to span the repeat regions. However, the sequencing of large fragments of nucleic acid is more difficult and time consuming in current sequencing apparatus.
Another approach to spanning a gap in the sequence is to determine the sequence of both ends of a large fragment. In contrast to single sequence reads of one end of a shotgun sequencing fragment, a pair of sequence reads from both ends have known spacing and orientation. The use of relatively long fragments also aids in the assembly of sequences containing interspersed repetitive elements. This type of approach (Smith, M. W. et al., Nature Genetics 7: 40-47 (1994) is known in the art as paired end sequencing. The present invention includes novel methods, systems and compositions useful for paired-end sequencing approaches and other nucleic acid technologies.